Komor ? Project Summary/Abstract - ?Development of New Genome Editing Agents Using RNA Modifying Enzymes? While targeted genome editing, the introduction of a specific modification in genomic DNA, has the potential to allow researchers to study and better understand mechanisms of human genetic diseases, traditional genome editing methods (including CRISPR-Cas9) that rely on the initial introduction of double stranded DNA breaks (DSB) suffer from modest genome editing efficiencies as well as unwanted gene alterations (indels), particularly when attempting to correct point mutations. Recently, a class of genome editing agents called single base editors was developed that does not involve DSBs, but rather uses a dCas9-tethered single-stranded DNA (ssDNA) modifying enzyme to directly chemically modify target nucleobases within a ~5 nucleotide window determined by the protospacer. Two classes of editors have been developed that use cytosine and adenine deamination chemistries to catalyze the conversion of C?G base pairs to T?A (CBEs), and A?T base pairs to G?C (ABEs), respectively. Here we propose the development and characterization of new base editors capable of facilitating new point mutations using methylation chemistry. We have use a bioinformatic approach to identify RNA modifying enzymes that have the potential to be repurposed into new base editors, and have rationally designed mutant libraries to use with directed evolution to convert these enzymes into base editors (Aim 1). Concurrently, we are developing a machine learning program that utilizes existing ssDNA modifying enzymes to identify putative mutations that will expand the substrate scope of the identified methyltransferases to ssDNA (Aim 2). Mutations identified from both strategies will then be tested and characterized for base editing in multiple orthogonal systems (Aim 3). The successful completion of the proposed work will represent a significant addition to existing base editing technologies, and will enable researchers to cleanly and efficiently install two additional types of point mutations into the genome of living cells, allowing researchers to quickly and effectively general model systems for the study of human genetic diseases.